Adoptive T cell therapy represents a promising strategy for the treatment of patients with cancer. Phase I and II studies using adoptively transferred antigen-specific T cell clones have led to the conclusion that its effectiveness may be enhanced by extending the in vivo persistence of transferred T cells and broadening the repertoire of immune responses to limit the outgrowth of antigen-loss tumor variants. Regulatory T cells (characterized by a CD25hi CD4 phenotype) are found at increased levels in patients with cancer, and may thwart an effective ant-tumor T cell response by suppressing T cell activation and effector function. We postulate that a pre-infusion conditioning regimen that decreases the regulatory T cell population will lead to extended in vivo persistence of adoptively transferred T cells and promote the generation of endogenous T cell responses against a broader panel of tumor-associated antigens. In this study we propose to examine the benefits of a combined biologic approach: adoptive transfer of antigen-specific CTL clones and CD25 lymphodepletion using DAB389-IL-2 (also known as denileukin diftitox or Ontak).We postulate that administration of DAB389-IL-2 to deplete regulatory T cells (Treg) might enhance not only the adoptively transferred T cell response, but also promote the generation of T cell responses against a broader panel of tumor-associated antigens that are released in the Treg-depleted pro-inflammatory environment following lysis of tumors by transferred antigen- specific CTL. We propose to evaluate the use of CD25 lymphodepletion as an adjunct to adoptive T cell therapy with the following Specific Aims: 1. Assess the safety and anti-tumor efficacy of cellular adoptive immunotherapy in melanoma patients using autologous CD8+ antigen-specific T cell clones following CD25 lymphodepletion 2. Determine the influence of CD25 lymphodepletion on the duration of in vivo persistence of adoptively transferred CD8+ antigen-specific CTL clones 3. Evaluate the induction of T cells to non-targeted tumor-associated antigens (antigen- spreading) following adoptive transfer of CD8+ antigen-specific CTL and CD25 lymphodepletion PUBLIC HEALTH RELEVANCE: Cancers that are resistant to standard chemotherapy and radiation may be treatable using components of the immune system. We propose to use immune cells, T cells that recognize targets on tumor cells as a means of treating patients with advanced (metastatic) melanoma. By isolating and expanding such T cells and infusing them into patients, we can track the survival and function of these cells and, we hope, identify a treatment approach that will be safe and will improve the effectiveness of melanoma- specific T cells.